The studies described in this proposal address the cellular mechanisms responsible for rapid modulation of hepatocyte ion transport to meet changing physiologic demands. In liver cells, dynamic interactions between cell metabolism, cell volume and ion transport play a key role in regulation of the response to circulating hormones, substrate availability, and metabolic stimuli. Previous studies indicate that this response is mediated in large part by a balance between channel-mediated Na+ influx, which drives cell volume increases, versus reciprocal K+ and Cl- efflux, which are responsible for cell volume decreases. Interestingly, preliminary observations indicate that; i) the distribution of ion channel proteins is carefully regulated between plasma membrane and intracellular membrane (vesicular) addresses, and ii) distinct pools of channel-containing vesicles can be mobilized rapidly and selectively to shape the conductive properties of the plasma membrane. Indeed, exocytosis of 10,000 or more vesicles is essential for the cellular response to hormones, and is sufficient to replace 20-40% of the plasma membrane within minutes. Accordingly, the Specific Aims are designed to address the following working hypothesis: regulated insertion and retrieval of distinct pools of channel-containing vesicles provides a mechanism for rapid modulation of the transport capacity of hepatocytes in response to changing physiologic demands, and represents an important site of action for hormones and other factors that utilize cell volume as an intermediary signal regulating cell metabolism and transport. The Specific Aims focus on 1) evaluation of non-selective cation (NSC) channels as the dominant mechanism for volume-regulatory Na + influx, 2) characterization of the cellular signals that couple volume-sensitive signaling to rapid exocytosis of distinct pools of channel-containing vesicles, and 3) assessment of a cDNA encoding cyclic nucleotide-gated (CNG) cation channels as a molecular candidate for the NSC conductance in liver cells. The interface between cell metabolism, ion channels, and cell volume represents a novel and powerful site of action for physiologic modulation of hepatic transport, secretion, and bile formation; and an important target for development of pharmacological approaches to modulation of liver cell and organ function.